Cryopreservation of human red blood cells

ABSTRACT

A red blood cell storage composition includes a composition of red blood cells and biochemistry altering reagents, the biochemistry altering reagents being present at a concentration so as to reduce the percent hemolysis of the red blood cells during the freeze-thaw cycle below that of the percent hemolysis of the red blood cells in the absence the biochemistry altering reagents. The red blood cell storage composition preferably includes reagents selected from: modifiers of glycolytic/metabolic components, modifiers of antioxidant potential, effectors of intracellular ionic distribution, modifiers of membrane fluidity, modifiers of cytoskeletal structure, effectors of the cyclooxygenase second messenger pathway, effectors of the lipoxygenase second messenger pathway, effectors of the hexose monophosphate second messenger pathway, effectors of the phosphorylation second messenger pathway, modifiers of specific messenger molecules, and combinations thereof.

Priority of provisional application Ser. No. 60/086,836 Filed: May 26,1998 is claimed.

BACKGROUND OF THE INVENTION

Blood is composed of plasma and cellular constituents and plays aprominent role in several key physiological systems of the human body,including immunology, hemostasis, and tissue oxygenation. Movement ofthe respiratory gases oxygen (O₂) and carbon dioxide (CO₂) is the chieffunction of erythrocytes or red blood cells. This facilitated movementOf O₂ and CO₂ is carried out by hemoglobin, an iron-containing,multi-subunit protein contained within red blood cells. The necessityfor encapsulating hemoglobin within the red blood cell is twofold.First, hemoglobin has very specific binding parameters to ensuredelivery of these critical molecules to the proper sites. By regulatingthe concentration of cofactors and free ions, the red blood cellprovides the proper environment for optimum uptake and release of O₂.Secondly, free heme has toxicity effects on both the renal and hepaticsystems and can lead to conditions such as hemoglobinuria.

The red blood cell structural components which confine the hemoglobinare comprised of the membrane bilayer and the cytoskeleton. The membraneitself contains choline and amino phospholipids, cholesterol, andintegral membrane proteins. Along the inner surface of the membrane liesthe cytoskeleton. This mesh-like skeleton is composed of long,filamentous spectrin molecules joined together at foci of F-actin andprotein 4.1 complexes. This cytoskeletal mesh is linked to the membranethrough protein-protein interactions such as the ankyrin/protein 4.2mediated connection of spectrin and the integral membrane protein Band3, and possibly via direct protein-lipid interactions. Through theseconnections, the cytoskeleton provides the red blood cell its stabilityand shape. In the absence of proper binding within the cytoskeletal meshor the connection of the cytoskeleton to the membrane, a criticallyweakened red blood cell results. Several human diseases have beenidentified stemming from either an absence of a specific protein or adefective protein interaction. Most of these defects lead tomorphologically abnormal red blood cells and severe hemolytic anemias.

A wide variety of injuries and medical procedures require thetransfusion of whole blood or a variety of blood components. Bloodtransfusions are routinely used to increase oxygen delivery capacity andcirculatory volume in patients. Safe, quick, and easy access totransfusable red blood cell units is not only important for traumavictims with massive blood loss, but also for patients undergoingelective surgery or who have diseases, such as several types ofhereditary hemolytic anemias, which result in a loss of circulating redblood cells. The ability to store red blood cells for extended timeperiods ensures that a supply of transfuable red blood cells will beavailable when needed. Potential uses include storage of uniqueserotypes, units intended for autologous transfusion and stockpilinggeneral blood types for emergency situations. Limitations in the currentstorage methodologies have led, in part, to occasional shortages ofblood supply, resulting in postponement of elective surgeries and callsfor donations from blood banks and hospitals.

Currently, two methods are approved for extended storage of red bloodcells. The majority of red blood cell units are stored in citratephosphate-dextrose at 4° C. For example, when donor blood is received ata processing center, erythrocytes are separated and stored by variousmethods, generally as a unit of packed erythrocytes having a volume offrom 200 to 300 ml and a hematocrit value of 70 to 90. However, due tometabolic depletion and subsequent physical degradation, these cells maybe stored no longer than 42 days. Furthermore, while 70% of these storedcells remain in circulation following transfusion, true O₂ deliverycapability has not been demonstrated.

During storage, human red blood cells undergo morphological andbiochemical changes, including decreases in the cellular level ofadenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG),changes in cellular morphology, and progressive hemolysis. Theconcentration of ATP, after a brief initial rise, progressively declinesto between 30 and 40% of its initial level after six weeks of storage.Morphological changes occur during storage, ultimately leading to thedevelopment of spicules which can bud off as vesicles, radicallychanging the surface-to-volume ratio of the cells and their ability todeform on passing through narrow channels. The fluidity of the cellmembrane of red cells, which is essential for the passage of red cellsthrough the narrow channels in the spleen and liver, is looselycorrelated with the level of ATP. The concentration of ATP and themorphology of red cells serve as indicators of the suitability of storedcells for transfusion.

The second method is frozen storage at −80° C. using 40% (w/v) glycerolas a cryoprotectant. However, this additive penetrates the membranes ofmany biological cells and possesses unwanted properties when infusedinto humans. While this method can be used for up to 10 years ofstorage, the glycerol must be washed out prior to transfusion. Thewashing procedure utilizes costly equipment and is time and laborintensive. Additionally, a significant level of hemolysis (10-15%typically) occurs during the wash procedure. Finally, since thisprocedure is also considered to compromise the sterility of the redblood cell units, post-wash storage is significantly limited.Consequently, this method of storage is only used for blood withextremely rare factor types and autologous and directed donation unitswhich will not be used within a 42 day period. As of 1992, the mostrecent data available, approximately 14% (nearly 2 million units) of theavailable supply of transfusable red blood cells were discarded. Theability to cryopreserve red blood cells without the added expense andtime of washing, or at least reducing the number of washing cyclesnecessary, would enable salvage of a significant percentage of thesediscarded units, thereby providing a further buffer against bloodproduct is shortages.

Current theories of red blood cell cryopreservation consider iceformation and propagation to be the primary, if not the sole factoraffecting cell recovery and viability following storage. Consequently,investigation of methods to cryopreserve red blood cells only considerslight variations of traditional cryoprotectant methodologies. Priormethods for the preservation, storage, and transfusion of red bloodcells are explained in Horn, Sputtek, Standl, Rudolf, Kuhnl, and Esch,Transfusion of Autologous, Hydroxyethyl Starch-Cryopreserved Red BloodCells, Anesth. Analg. 1997; 85; 739-745. However, temperature-inducedmodulation of cellular biochemistry is a well recognized phenomenon byexperts in this field. The descriptions herein provide methods ofutilizing the red blood cell biochemistry to overcome cold-inducedimbalances which would normally lead to cellular hemolysis followingcryopreservation. Through these methods, the level of non-specificcryoprotection against ice formation may be reduced.

Two general procedures are currently available for the frozenpreservation of living cells in terms of the cryoprotectant used. Oneutilizes the addition of high concentrations of low molecular weightpenetrant solutes. Due to toxicity and osmotic problems, specializedequipment and techniques are required to remove the solutes followingthawing. The second utilizes high molecular weight polymers which do notenter the cells. Post-thaw processing in this case is logisticallysimpler but storage must be at very low temperature, typically in liquidnitrogen. The formation of ice, which is a prime concern in any methodof cell cryopreservation, is initiated by ice crystal nuclei. As thetemperature falls and water crystallizes to ice, the cell isprogressively dehydrated and at some point cell injury results. Thenature of the dehydration injury is believed to be the result ofmembrane stresses leading to membrane rupture. This form of injury canbe prevented by the addition of solutes at a multi-molar concentrationso that the amount of ice formed is insufficient to result in damagingcell dehydration.

For many years it has been known that certain molecules may becryoprotective. It is necessary for these penetrating cryoprotectants toreduce the amount of extracellular ice formed and thereby reduce celldehydration, while at the same time increasing the intracellularconcentration to make intracellular crystallization less likely. Such asolute, to be useful, must is be non-toxic at high concentration andmust freely penetrate the cell. Both glycerol and dimethylsulfoxide(DMSO) have been used for this purpose. Glycerol is remarkably non-toxicat high concentrations but penetrates cell membranes slowly and istherefore difficult to introduce and remove. Dimethylsulfoxidepenetrates rapidly but becomes increasingly toxic as concentrationsexceed 1 M (about 7%). Large molecular weight polymers, such aspolyvinylpyrrolidone (PVP), dextran, and more recently hydroxyethylstarch (HES), do not enter the cell, and the mechanism by which theyconfer cryoprotection has been the subject of speculation.

As stated above, in order to prolong the storage of red blood cells itis necessary to store the cells or treat them in some manner thatprevents a decline in ATP, and, if possible, 2,3-diphosphoglycerate(2,3-DPG), in addition to protection against ice crystal damage.Typically, such solutions contain phosphate, glucose, and adenine whichfunction to prolong shelf-life by maintaining the level of ATP in thecells. In addition, glycolytic activity is enhanced in red blood cellsif the intracellular pH measured at 4° C. is about 7.4. The effectiveosmolality of the suspending solution is another factor of importance inextending red cell storage time. It has been shown that hypotonicallyinduced increases in mean cell volume substantially reduce hemolysis andimproves red cell morphology during storage. Although the mechanism hasnot been proven, it is possible that osmotic swelling increases cellsurface tension, thereby opposing the shape changes usually associatedwith stored red cells. Although the hypotonicity of the additivesolution is limited by the danger of hemolysis during the addition ofthe solution, red cells, which are normally bi-concave disks, can swellto nearly twice their normal volume at an external osmolality ofapproximately 170 mOsm before they hemolyze. If the additive solution istoo hypotonic, the red cells will burst (hemolyze). As a result,solutions that are too hypotonic cannot be used. While maintenance ofATP and 2,3-DPG are generally thought of with reference to 4° C.storage, maintaining the concentration of these metabolites is importantfor post-thaw storage. For the effects on ATP levels, hemolysis,potassium leakage, and shedding of microvesicles, on the maintenance andstorage of red blood cells, see Greenwalt, Rugg, and Dumaswala, TheEffect of Hypotonicity Glutamine, and Glycine on Red Cell Preservation,Transfusion 1997; 37; 269-276.

The goal of extended red blood cell storage is to maintain the metabolicand morphologic characteristics so that the in vivo parameters (oxygendelivery capacity and circulatory half-life) are comparable to fresh,non-frozen red blood cells. Additionally, the level of hemolysis duringthe storage cycle must remain within acceptable levels to allow directtransfusion of the thawed red blood cell unit without complications fromhemoglobin toxicity. These goals can be accomplished using the method ofthis invention. The method described herein provides the advantages ofboth currently approved storage methods—extended storage time in thefrozen state and immediate availability as in refrigerated storage.

Hospitals and blood banks would greatly benefit from adirectly-transfusable frozen red blood cell product. Most currentinvestigational approaches to accomplish this attempt to merely replaceglycerol with a non-toxic, transfusable cryoprotectant. However, resultshave shown that most cryoprotectants need to be used at concentrationswhich may not be transfused or their protective qualities are not enoughto maintain hemolysis at acceptable levels for transfusion.Additionally, most procedures require storage at −193° C. in liquidnitrogen vapor. This would be expensive and difficult to incorporateinto current blood banking procedures, making extended storage attemperatures below −80° C. impractical.

The invention described herein addresses the noted problems of storage,morphological changes, metabolic changes, and long term functionaleffectiveness of red blood cells. Further, the present inventionachieves unexpected and surprising results in the preservation of redblood cells through treating with the compositions and methods of thisinvention and which previously would have been considered impossible.

SUMMARY OF THE INVENTION

This invention provides a method for storing red blood cells in thefrozen state at subzero temperatures (−10° to −193° C.) such that theresulting thawed red blood cell unit contains viable cells.Specifically, this is accomplished through a combination of biochemicalstabilization and cryoprotectant solution. A biochemical stabilizingreagent is one that provides no traditional cryoprotection, in that italone does not diminish the formation or quantity of ice during thefreezing process at the concentration used. In the event that thecombined cryoprotectant is solution is composed of transfusablecryoprotective reagents, the resulting thawed red blood cell unit isdirectly transfusable. In all circumstances, incorporating biochemicalstabilizing technology allows cryopreservation of red blood cellsutilizing concentrations of cryoprotectants lower than would benecessary in the absence of biochemical stabilization.

Biochemical stabilization involves the addition of reagents which targetspecific red blood cell components and biochemical pathways susceptibleto damage during the freeze/thaw cycle. Stabilization with thesereagents renders the cells partially resistant to freeze/thaw-induceddamages, which ultimately results in hemolysis. Biochemicalstabilization may be targeted to maintain metabolic components,antioxidant potential, intracellular ionic distribution, membranefluidity, and integrity of the cytoskeletal structure. Additionally,specific second messenger pathways, such as the cyclooxygenase,lipoxygenase, hexose monophosphate and phosphorylation pathways, may bemanipulated directly by biochemical reagents or indirectly throughregulation of specific messenger molecules, including cyclic-adenosinemonophosphate (c-AMP), cyclic-guanine monophosphate (c-GMP),intracellular calcium, inositol triphosphate, and diacylglycerol.

More specifically, a single reagent or combination of reagents may beadded to red blood cell units to target each of the above items. Forexample, reagents can be added to maintain or enhance intracellularconcentrations of ATP such as glucose, pyruvate or inorganic phosphateas well as blocking ATP-depleting ATPases such as amiloride mediatedinhibition of the Na⁺/H⁺ exchanger. Preventing oxidative damage to themembrane or hemoglobin may be accomplished through the addition ofantioxidants such as glutathione, tocopherol, ascorbate andbioflavonoids as well as through the stimulation of the hexosemonophosphate pathway by ribose. Ionic distribution can be regulatedthrough activation or inhibition of specific ion pumps. Calcium may becontrolled with nifedipine or verapamil while amiloride will effectsodium regulation as mentioned above. Further potassium and chlorideionic distribution may be maintained through inhibition of the K⁺/Cl⁻co-transporter with bumetamide. The cyclooxygenase and lipoxygenasepathways may be regulated through the addition of flurbiprofen,dipyridamole, or aspirin while phosphorylation events can be manipulatedthrough inhibition by kinase inhibitors such as H7(1-[5-isoquinolinylsulfonyl]-2-methylpiperazine), staurosporin, orchelerythrine or stimulated by diacylglycerol palmitoyl carnitine.Membrane stabilization and fluidity is controlled through the additionof pentoxifylline, nicotinamide or amantadine and cytoskeletal structuremay be maintained through actin stabilization by the use of Taxol,[2aR-[2aα,4β,4aβ,6β,9α(αR*,βS*,11α,−12α,12aα,12bα]]-β-(Benzoylamino)-α-hydroxybenzenepropanoic acid6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,-13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz-[1,2-b]oxet-9-yl-ester, or cytochalasins or spectrinstabilization with polyamines. Specific cyclic nucleotide regulatedsecond messenger pathways may be activated through the use of adenosine(elevates c-AMP) and sodium nitroprusside or other nitric oxide donors(elevates c-GMP).

In addition to biochemical stabilization, cryoprotectants are used toprotect the red blood cell unit from damage by ice crystal formationduring the freeze/thaw cycle. The cryoprotectants may be usedindividually or as mixtures made up of penetrating and/ornon-penetrating compounds. Examples of potential cryoprotectantsinclude, but are not limited to dimethylsulfoxide, polyvinylpyrrolidone, dextran, maltodextrins, 2,3-butanediol, hydroxyethylstarch, polyethylene glycol and glucose and other carbohydrates.

In one embodiment for preserving human red blood cells the methodincludes drawing a volume of packed red blood cells in ADSOL, acurrently licensed additive solution sold by Baxter Travenol, usingstandard blood banking protocols. The ADSOL is removed bycentriftigation, and a volume of preservation solution containing thedesired cryoprotectants and any additional biochemical reagents isadded. The red blood samples containing cryoprotectants are submerged inliquid nitrogen for freezing and immediately transferred to a −80° C.freezer for extended storage. Biochemical stabilization may be targetedto maintain metabolic components, antioxidant potential, intracellularionic distribution, membrane fluidity and integrity of cytoskeletalstructure. Additionally, specific second messenger pathways, such as thecyclooxygenase, lipoxygenase, hexose monophosphate and phosphorylationpathways, may be manipulated directly by biochemical reagents orindirectly through regulation of specific messenger molecules, includingc-AMP, c-GMP, intracellular calcium, inositol triphosphate, anddiacylglycerol. In one preferred embodiment the biochemical reagents arepresent in a concentration so as to prevent the hemolysis of the redblood cells during the freeze/thaw cycle and even more preferably haveconcentrations of about 500 μM nifefipine, about 20 μM cytochalasin B,about 500 μM Taxol, about 5 mM pentoxifylline, and about 25 μg/mlflurbiprofen. The biochemical reagents are present in 2.5% DMSO as acarrier agent. All conditions contain 7.5% dextran (Dex; 40,000 MW), 2%polyvinyl pyrrolidone (PVP; 40,000 MW), 5% hydroxyethyl starch, and 5%polyethylene glycol. The low, transfusable concentrations ofcryoprotectants can work in combination to protect the red blood cellsduring cryopreservation better than any single cryoprotectant alone.Moreover, the addition of the reagents and the biochemical modulationwith the low concentrations of transfusable cryoprotectants works incombination to further protect the red blood cells duringcryopreservation. An alternative method is to add the isotonic saline orpreservative solution containing the biochemical reagents and a volumeof Glycerolyte 57 (Fenwall), preferably about 20% glycerol and freezethe bags at −80° C. The additives used include nicotinamide,nikethamide, nifedipine, pentoxifylline, and flurbiprofen. The 20%glycerol with the addition of the biochemical additives reduces thelevel of hemolysis and meets the criteria for a transfusable blood unitbased on the overall hemolysis and residual free hemoglobin levels.

Thus, one embodiment of the present invention is a red blood cellstorage composition including a red blood cell composition and acomposition of biochemical stabilization reagents. In such an embodimentthe biochemical reagents should be present at a concentration so as topermit decreased hemolysis during the freeze-thaw cycle and increased invitro functional activity when compared to red blood cells preservedunder the same conditions but in the absence of the biochemicalreagents.

In yet another embodiment of the present invention, a red blood cellcomposition is formed comprising red blood cells, a composition of redblood cells and biochemical reagents, and a combination of one or morecryoprotective agents.

Another embodiment is directed to a human red blood cell compositioncomprising human red blood cells, a composition of red blood cells andbiochemical reagents. Reagents can be added to maintain or enhanceintracellular concentrations of ATP such as glucose, pyruvate orinorganic phosphate as well as blocking ATP-depleting ATPases such asamiloride inhibition of the Na⁺/H⁺ exchanger. Preventing oxidativedamage to the membrane or hemoglobin may be accomplished through theaddition of antioxidants such as glutathione, tocopherol, ascorbate andbioflavonoids as well as through the stimulation of the hexosemonophosphate pathway by ribose. Ionic distribution can be regulatedthrough activation or inhibition of specific ion pumps. Calcium may becontrolled with nifedipine or verapamil while amiloride will effectsodium regulation as mentioned above. Further potassium and chlorideionic distribution may be maintained through inhibition of the K⁺/Cl⁻co-transporter with bumetamide. The cyclooxygenase and lipoxygenasepathways may be regulated through the addition of flurbiprofen,dipyridamole, or aspirin while phosphorylation events can be manipulatedthrough inhibition by kinase inhibitors such as H7(1-[5-isoquinolinylsulfonyl]-2-methylpiperazine), staurosporin, orchelerythrine or stimulated by diacylglycerol palmitoyl carnitine.Membrane stabilization and fluidity is controlled through the additionof pentoxifylline, nicotinamide or amantadine and cytoskeletal structuremay be maintained through actin stabilization by the use of TAXOL orcytochalasins or spectrin stabilization with polyamines. Specific cyclicnucleotide regulated second messenger pathways may be activated throughthe use of adenosine (elevates c-AMP) and sodium nitroprusside or othernitric oxide donors (elevates c-GMP). The biochemical reagents arepresent in a concentration so as to prevent the hemolysis of the redblood cells during the freeze/thaw cycle.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The biochemical stabilization of this invention is based on theapplication of specific effectors, which target specific aspects ofcellular biochemistry to protect cells against specific modes of damage.The method of cryopreservation is such that the stored cells may bedirectly transfused following thaw, by combining biochemical reagentsand low concentrations of transfusable cryoprotectants. The biochemicalstabilization involves regulation of the metabolic, ionic, cytoskeletal,and membrane component, rendering the treated red blood cells lesssusceptible to damage induced during freezing and thawing attemperatures between −10° C. to −193° C.

In one embodiment, specific cellular effectors are nifedipine,cytochalasin B, Taxol, pentoxifylline, flurbiprofen, nikethamide,ribose, and trehalose. These modifiers are added to the packed red bloodcells and the preservative solution. Each of the modifiers affectsdifferent specific cellular biochemistry. In one aspect of the presentinvention, nifedipine an inhibitor acting through the calcium cascadecontrols calcium, while, cytochalasin B and Taxol are cytoskeletalmodifiers and provide stabilization to the cell through actinstabilization. In another aspect of the present invention, membranestabilization and fluidity may be controlled through pentoxifylline, amembrane modifier. Ribose is added to prevent oxidative damage to thecell by stimulation of the hexose monophosphate pathway.

The second messenger effectors have been demonstrated to providebiochemical stabilization to the red blood cells either individually orin combination with others. More, importantly, the addition of thesereagents allows biochemical modulation to enhance cryoprotection atlower glycerol concentrations which normally result in nearly 100% ofthe red blood cells being inadequately protected during the freeze/thawand washing cycles. In describing the chemicals which have shown utilityas effectors of the second messenger pathways, it must be understoodthat the actual chemicals mentioned together with functionallyequivalent materials are intended to be within the scope of thisinvention. Chemicals that are known to applicants to have known ordemonstrated utility as modifiers have been specifically set forth inthe instant application. However, it is intended that the scope of theapplication be extended to other functionally effective chemicals, bothexisting chemicals and chemicals yet to be discovered.

Certain chemicals which are thought to be functionally equivalentmaterials for the modifier acting through the second messenger pathwayare those selected from cyclic-adenosine monophosphate, c-(AMP),cyclic-guanine monophosphate, c-(GMP), intracellular calcium, inositoltriphosphate, and diacylglycerol. Functionally equivalent modifiersacting through the cyclic nucleotide regulated second messenger pathwayare those selected from adenosine, sodium nitroprusside, and othernitric oxide donors. Materials thought to be functionally equivalent tothe modifier of intracellular concentrations of ATP are those selectedfrom glucose, pyruvate, and inorganic phosphate, as well asATP-depleting ATPases such as amiloride. Preventing oxidative damage tothe membrane or hemoglobin may be accomplished through the addition offunctionally equivalent antioxidants such as glutathione, tocopherol,ascorbate and bioflavonoids as well as through the stimulation of thehexose monophosphate pathway by ribose. Ionic distribution can beregulated through activation or inhibition of specific ion pumps.Functionally equivalent modifiers of calcium may be selected fromnifedipine or verapamil while amiloride will effect sodium regulation asmentioned above. Further potassium and chloride ionic distribution maybe maintained through inhibition of the K⁺/Cl⁻ co-transporter withbumetamide. The cyclooxygenase and lipoxygenase pathways may beregulated through the addition of functionally equivalent modifiers offlurbiprofen, dipyridamole, or aspirin while phosphorylation events canbe manipulated through inhibition by kinase inhibitors such as H7(1-[5-isoquinolinylsulfonyl]-2-methylpiperazine), staurosporin, orchelerythrine or stimulated by diacylglycerol palmitoyl carnitine, whichare also functionally equivalent reagents. Reagents which arefunctionally equivalent regulators of membrane stabilization andfluidity are pentoxifylline, nicotinamide or amantadine, while for theregulation of cytoskeletal structure through actin stabilization, thereagents are TAXOL or cytochalasins and for spectrin stabilization,polyamines. Specific cyclic nucleotide regulated second messengerpathways may be activated through the use of functionally equivalentadenosine (elevates c-AMP) and sodium nitroprusside or other nitricoxide donors (elevates c-GMP).

The post-thaw life of the red blood cells after cryopreservation may besuccessfully extended by storing the cells at −80° C. with thebiochemical reagents of this invention. When red blood cells were storedfor 3-7 days in a −80° C. freezer and thawed at 37° C. in a water bathand analyzed for post-storage hemolysis, as compared to red cells storedin glycerol concentrations alone, the percentage of hemolysis was asfollows: 14.36% with nifedipine, 15.01% with Taxol and cytochalasin B,11.46% with nifedipine, pentoxifylline, and flurbiprofen, and 11.66%with nifedipine, cytochalasin, and Taxol. These results comparefavorably to enhance cryoprotection when compare to glycerolconcentrations which normally result in nearly 100% of the red bloodcells being inadequately protected during the freeze/thaw and washingcycles.

To perform the experiment, a packed red blood cell unit in ADSOL wasobtained using standard blood banking protocols. The ADSOL was removedby centrifugation and a volume of preservation solution containing thedesired cryoprotectants and biochemical reagents was added. The finalconcentrations of cryoprotectant used were 7.5% dextran, 2% polyvinylpyrrolidone, 5% hydroxyethyl starch, and 2.5% dimethyl sulfoxide as abiochemical reagent carrier. Reagents used included 500 μM nifedipine,20 μM cytochalasin B, 500 μM Taxol, 5 mM pentoxifylline and 25 μg/mlflurbiprofen. After the ADSOL was removed and the preservation solutionwas added, 50 ml of red cells was placed into freezing bags having amaximum volume of 150 ml. An equal volume was added to each of the bags.The bags were plunge frozen and stored at −80° C. for 2-7 days andthawed by submersion a 37° C. water bath for 10 minutes. Level of thawhemolysis was determined as the ratio of free hemoglobin to totalhemoglobin. This biochemical reagent mixture is able to be directlytransfused following storage.

Storing red blood cells at −10° C. to −193° C. requires the addition ofa cryoprotective agent, such as dimethlyl sulfoxide (DMSO).Cryoprotective agents such as DMSO are polar molecules which penetratesthe cell membrane and serves to preserve cell viability during thecryopreservation process. In addition to DMSO other cryoprotectiveagents used in this invention include polyvinyl pyrrolidone, dextranmaltodextrins, 2,3-butanadiol, hydroxyethyl starch, polyethylene glycol,glucose, and combinations thereof. The success of cryopreservation withcertain water soluble molecules which do not permeate the cells has beenreported, however, the exact mechanism for this success is unknown. Ithas been speculated that this phenomenon including that the polymersprotect cells by lowering extracellular salt concentrations atsubfreezing temperatures just as penetrating cryoprotectants do, or,that the polymers might adsorb to cells and thus protect the membrane insome way. It has also been speculated that during freezing anelectrolyte gradient develops from inside to outside the cells causingan electrolyte leakage which relieves osmotic stress. Thecryoprotectants may be used individually or as mixtures made up ofpenetrating and/or non-penetrating compounds in transfusableconcentrations to protect the red blood cells from damage by ice crystalformation during the freeze/thaw cycle.

For example, it can be shown by a comparison of the followingpercentages of thaw hemolysis that combinations of transfusableconcentrations of cryoprotectants allow increased protection fromhemolysis than a single cryoprotective agent: 7.5% dextran, 11.19%hemolysis; 2% polyvinyl pyrrolidone, 47.39% hemolysis; 5% hydroxyethylstarch, 43.17% hemolysis; 7.5% dextran and 2% polyvinyl pyrrolidone,6.42% hemolysis; 7.5% dextran and 5% hydroxyethyl starch, 8.56%hemolysis; 2% polyvinyl pyrrolidone and 5% hydoxyethyl starch, 23.46% ishemolysis, and finally 7.5% dextran, 2% polyvinyl pyrrolidone, and 5%hydroxethyl starch, 4.44% hemolysis.

A red blood cell storage composition of the present invention, includesa storage composition, a composition of red blood cells, and acomposition of biochemical reagents, the reagents present in aconcentration so as to prevent the hemolysis of the red blood cellsduring the freeze/thaw cycle and in a concentration to increase the invitro functional activity when compared to red blood cells preservedunder the same conditions but in the absence of the biochemicalreagents. As the term is used herein and in the claims the “storagecomposition,” is intended to mean a pharmacologically inert fluid intowhich the red blood cells may be suspended and which does not adverselyaffect the preservation abilities of the compositions disclosed herein,for example physiological saline. Biochemical stabilization may betargeted to maintain metabolic components, antioxidant potential,intracellular ionic distribution, membrane fluidity and integrity ofcytoskeletal structure. Additionally, specific second messengerpathways, such as the cyclooxygenase, lipoxygenase, hexose monophosphateand phosphorylation pathways, may be manipulated directly by biochemicalreagents or indirectly through regulation of specific messengermolecules, including cyclic-adenosine monophosphate, (c-AMP),cyclic-guanine monophosphate, (c-GMP), intracellular calcium, inositoltriphosphate, and diacylglycerol.

In one preferred embodiment the modifiers acting through the secondmessenger pathway is selected from cyclic-adenosine monophosphate,(c-AMP), cyclic guanine monophosphate, (c-GMP), intracelluar calcium,inositol triphosphate, and diacylglycerol. Reagents can be added tomaintain or enhance intracellular concentrations of ATP such as glucose,pyruvate or inorganic phosphate as well as blocking ATP-depletingATPases such as amiloride inhibition of the Na⁺/H⁺ exchanger. Preventingoxidative damage to the membrane or hemoglobin may be accomplishedthrough the addition of antioxidants such as glutathione, tocopherol,ascorbate and bioflavonoids as well as through the stimulation of thehexose monophosphate pathway by ribose. Ionic distribution can beregulated through activation or inhibition of specific ion pumps.Calcium may be controlled with nifedipine or verapamil while amiloridewill effect sodium regulation as mentioned above. Further potassium andchloride ionic distribution may be maintained through inhibition of theK⁺/Cl⁻ co-transporter with bumetamide. The cyclooxygenase andlipoxygenase pathways may be regulated through the addition offlurbiprofen, dipyridamole, or aspirin while phosphorylation events canbe manipulated through inhibition by kinase inhibitors such as H7(1-[5-isoquinolinylsulfonyl]-2-methylpiperazine), staurosporin, orchelerythrine or stimulated by diacylglycerol palmitoyl carnitine.Membrane stabilization and fluidity is controlled through the additionof pentoxifylline, nicotinamide or amantadine and cytoskeletal structuremay be maintained through actin stabilization by the use of TAXOL orcytochalasins or spectrin stabilization with polyamines. Specific cyclicnucleotide regulated second messenger pathways may be activated throughthe use of adenosine (elevates c-AMP) and sodium nitroprusside or othernitric oxide donors (elevates c-GMP).

In another preferred embodiment, the biochemical reagents are:nifedipine in a concentration of about 500 μM, cytochalasin B in aconcentration of about 20 μM, Taxol in a concentration of about 500 μM,pentoxifylline in a concentration of about 5 mM, and flurbiprofen in aconcentration of about 25 μg/ml. The blood cell storage composition ofthe present embodiment may further include a cryoprotective agent, thecryoprotective agent being selected from dimethyl sulfoxide, polyvinylpyrrolidone, dextran maltodextrins, 2,3-butandiol, hydroxyethyl starch,polyethylene glycol, glucose, and combinations thereof. Preferably, thecryoprotective agents are: dextran in a concentration of about 7.5%;polyvinyl pyrrolidone in a concentration of about 2%; dimethyl sulfoxidein a concentration of about 2.5%; hydroxyethyl starch in a concentrationof about 5%, and polyethylene glycol in a concentration of about 5%. Theprolonged storage of the red blood cells should be carried out at atemperature below the red cells' normal physiological temperature. Inone embodiment, the temperature is from about —10° C. to about −193° C.In another embodiment the temperature below the red blood cells' normalphysiological temperature is about −80° C.

The following examples demonstrate the ability of biochemicalstabilization to enhance the preservation of red blood cells attemperatures less than −10° C. Both glycerol-based and transfusable,polymeric-based cryoprotectant solutions are described.

The examples are included to demonstrate preferred embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept and scope ofthe invention.

EXAMPLE 1

The following example demonstrates the damage induced during thefreeze/thaw cycle when the glycerol concentration is decreased. Asignificant percentage of the hemolytic damage is shown to be reversedby the addition of a preservative solution containing adenine,glutamine, sodium chloride, mannitol, and dextrose, with the essentialadditive being dextrose, and final concentrations of 5 mM pentoxifyllineand 25 μg/ml flurbiprofen in DMSO as a carrier.

Specifically, 50 ml of packed red blood cells was added to a PVCfreezing bag with a maximum volume of 150 ml. Isotonic saline orpreservative solution containing the additional biochemical reagentspentoxifylline and flurbiprofen was mixed with the red blood cells. Avolume of Glycerolyte 57 (Fenwall) was added slowly until the finalconcentration of glycerol was achieved. The total volume of alladditions was 50 ml, making the final concentration of the red bloodcell unit 100 ml. The PVC bags were frozen at −80° C. for 2-7 days andthawed by submersion in a 37° C. water bath for 10 minutes. The level ofthaw hemolysis was determined as the ratio of free hemoglobin to totalhemoglobin. The levels of thaw hemolysis are shown below in TABLE 1.TABLE 1 LEVELS OF THAW HEMOLYSIS % Hemolysis 40% Glycerol 1.91 30%Glycerol 3.53 20% Glycerol 35.29 20% + Additives 14.87

These results confirm the loss of red blood cell protection at reducedglycerol concentrations and the ability of biochemical reagents toprovide cryoprotection at glycerol concentrations which alone are unableto completely protect red blood cells during cryopreservation.

EXAMPLE 2

The following example further demonstrates the damage induced during thefreeze/thaw cycle when the glycerol concentration is decreased, afterred blood cells are frozen and stored at −80° C. Fifty ml of packed redblood cells were added to a PVC freezing bag. Isotonic saline orpreservative solution containing the additional biochemical reagents ofinterest was mixed with the red blood cells. The samples were treated asfollows prior to freezing at −80° C.:

1. 40% Glycerol

2. 20% Glycerol

3. Nicotinamide

4. Nikethamide, Nifedipine

5. Nikethamide, Nifedipine, Pentoxifylline, Flurbiprofen

6. Nicotinamide Nikethamide, Nifedipine, Pentoxifylline, Flurbiprofen

A volume of Glycerolyte 57 (Fenwall) was added slowly until the finalconcentration of glycerol was achieved. The total volume of alladditions was 50 ml, making the final volume of the red blood cell unit100 ml. The PVC bags were frozen at −80° C. for 2-7 days and thawed bysubmersion in a 37° C. water bath for 10 minutes. The level of thawhemolysis was determined as the ratio of free hemoglobin to totalhemoglobin. Additionally, the glycerol was removed from the thawed redblood cell units by serial dilution with saline similar to blood bankprotocols. The overall hemolysis and the final residual free hemoglobinlevels were also determined.

TABLE 2 compares 40% glycerol to 20% glycerol and 20% glycerol with theindicated additives. The additives used included nicotinamide,nikethamide, nifedipine, pentoxifylline, and flurbiprofen. The resultsof 20% glycerol with all additives meet the criteria for a transfusableblood unit based on the overall hemolysis and residual free hemoglobinlevels. These results confirm the loss of red blood cell protection atreduced glycerol concentrations and the ability of biochemical reagentsto provide cryoprotection at glycerol concentrations which alone areunable to completely protect red blood cell during cryopreservation.TABLE 2 Biochemical Stabilization with Glycerol Based CryoprotectantHemolysis (%) Condition Thaw Overall Final 40% Glycerol 2.01 14.1 0.4520% Glycerol 9.39 41.48 1.09 Nicotinamide 8.13 22.93 0.43Nikethamide/Nifedipine 9.65 37.23 0.8 Nikethamide/Nifedipine 9.94 34.480.69 Pentoxifylline/Flurbiprofen Nicotinamide 9.07 17.84 0.33Nikethamide/Nifedipine Pentoxifylline/Flurbiprofen

EXAMPLE 3

The following experiment demonstrates the ability of biochemicaladdition to provide protection against damage during cryopreservation.The model used is a reduced glycerol cryopreservation system. 10% (w/v)glycerol is added to the unit prior to freezing at −80° C. A significantlevel of hemolysis occurs so that protection by the added reagent may bedistinguished.

A single unit of packed red blood cells in ADSOL was obtained usingstandard blood banking protocols. The ADSOL was removed bycentrifugation and a volume of preservation solution was added until theresulting hematocrit was about 50%. From this red blood cell unit 50 mlof red cells was placed into each of 3 PVC freezing bags having amaximum volume of 150 ml. An equal volume (50 ml) was added to each ofthe bags composed of the above preservation solution and theexperimental conditions. The final composition of the first bag was 10%(w/v) glycerol. The second contained 10% (w/v) glycerol and 2.5% DMSOand 500 μM nifedipine. The bags were frozen at −80° C. for 2-7 days andthawed by submersion in a 37° C. water bath for 10 minutes. The level ofthaw hemolysis was determined as the ratio of free hemoglobin to totalhemoglobin. Additionally, the glycerol was removed from the thawed redblood cell units by serial dilution with saline similar to blood bankprotocols. The overall hemolysis was also determined. The results areshown below in TABLE 3. TABLE 3 OVERALL HEMOLYSIS % Hemolysis ThawOverall 10% Glycerol 61.71 ± 1.26 93.62 ± 1.28 w/2.5% DMSO 59.45 ± 0.9081.86 ± 2.72 w/500 μM Nifedipine 55.12 ± 1.58 70.94 ± 2.96

This example further confirms the ability of biochemical modulation toenhance cryoprotection at glycerol concentrations which normally resultin nearly 100% of the red blood cells being inadequately protectedduring the freeze/thaw cycles.

EXAMPLE 4

This example demonstrates the ability of biochemical stabilization toenhance red blood cell cryopreservation in conjunction with a glycerolbased cryoprotectant. An aliquot of blood was transferred to a PVCstorage bag, to which any experimental agents were added. Glycerol wasthen added until the desired final glycerol was achieved. This unit wasfrozen at −80° C., stored and thawed in a 37° C. water bath for 10minutes. The glycerol was removed by serial dilution and centrifugationwith 12%, 1.6% and 0.9% saline until the residual free hemoglobin isless than 2% of the total hemoglobin content.

TABLE 4 show results following the desired procedure. The valuesindicate the percent hemolysis in the thawed sample, the overall amountof hemolysis following the wash and the is percent of residual freehemoglobin in the final sample. Hemolysis is calculated as the ratio offree hemoglobin to the total hemoglobin content in the sample. TABLE 4BIOMEDICAL STABILIZATION WITH GLYCEROL-BASED CRYOPROTECTANT % HemolysisCondition Thaw Overall 40% Glycerol 1.9 6.1 20% Glycerol with Control37.9 52.0 20% Glycerol with 20.6 27.6 Pentoxifylline 20% Glycerol with20.2 38.5 Nifedipine 20% Glycerol with 23.6 58.7 Amiloride 20% Glycerolwith 22.9 46.9 Flurbiprofen 20% Glycerol with 12.5 19.3 PentoxifyllineFlurbiprofen 20% Glycerol with 11.0 17.0 Nikethamide 20% Glycerol with4.2 7.8 Nicotinamide 20% Glycerol with 7.8 14.0 Nikethamide NifedipineNicotinamide

EXAMPLE 5

The following example demonstrates that biochemical stabilization usingnon-cryoprotective reagents reduces the level of hemolysis when usingalternative polymeric reagents as the primary cryoprotectants, when redblood are plunge frozen and subsequently stored at −80° C. A volume ofpacked red blood cell was mixed with an equal volume of preservationsolution containing the desired cryoprotectants and any additionalbiochemical reagents. The final concentrations of cryoprotectants usedwere 7.5% dextran (Dex; 40,000 MW) and 2% polyvinyl pyrrolidone (PVP;40,000 MW). The red blood cell samples containing cryoprotectants weresubmerged in liquid nitrogen for 3 minutes to freeze and immediatelytransferred to a −80° C. freezer for extended storage. Following storagefor 3-7 days, the samples were thawed at 37° C. in a water bath, and thelevel of thaw hemolysis was determined as the ratio of free hemoglobinto total hemoglobin. Additionally, the glycerol was removed from thethawed red blood cell units by serial dilution with saline similar toblood bank protocols. The overall hemolysis and the final residual freehemoglobin were also determined.

TABLE 5 shows the biochemical stabilization with a polymeric basedcryoprotectant and the resulting percent hemolysis. All samplescontained 7.5% Dex and 2% PVP, 2.5% dimethyl sulfoxide as a biochemicalcarrier, prior to storage at −80° C., in addition to the indicatedadditive as follows:

1. DMSO

2. Nifedipine

3. Cytochalasin, Taxol

4. Nifedipine, Pentoxifylline, Flurbiprofen

5. Nifedipine, Cytochalasin, Taxol

Reagents used include 500 μM nifedipine, 20 μM cytochalasin B, 500 μMTaxol, 5 mM pentoxifylline, and 25 μg/ml flurbiprofen in severalcombinations.

This example further confirms the ability of biochemical modulation toenhance cryoprotection at glycerol concentrations which normally resultin nearly 100% of the red blood cell being inadequately protected duringthe freeze/thaw and washing cycles. The biochemical stabilization with apolymeric cryoprotectant and resulting percentage hemolysis is displayedin TABLE 5. TABLE 5 Biochemical Stabilization with Polymeric BasedCryoprotectant Condition % Hemolysis DMSO 23.17 ± 0.58 Nifedipine 14.36± 0.45 Cytochalasin/Taxol 15.01 ± .70  Nifedipine 11.46 ± 0.44Pentoxifylline/Flurbiprofen Nifedipine/Cytochalasin/Taxol 11.66 ± 0.37

EXAMPLE 6

The following example describes an experiment to measure the biochemicalstabilization and the post-thaw hemolytic lesion upon dilution,analogous to transfusion, after red blood cell are plunge frozen andstored at −80° C. This type of storage lesion is not commonly addressedbut is observed with most non-glycerol based cryoprotective solutions. Avolume of packed red is blood cell was mixed with an equal volume ofpreservation solution containing the desired cryoprotectants and anyadditional biochemical reagents. Using a cryoprotective additivesolution (CPA) containing 7.5% dextran (Dex; 40.000 MW) 2% polyvinylpyrrolidone (PVP; 40,000 MW), 5% hydroxyethyl starch (HES), and 5%polyethylene glycol (PEG), the red blood cell samples containingcryoprotectants were submerged in liquid nitrogen for 3 minutes tofreeze and immediately transferred to a −80° C. freezer for extendedstorage. Following storage the thaw samples were split into twoaliquots. The first aliquot was analyzed immediately, and the remainingaliquot was incubated at 4° C. for 3 hours prior to analysis. Theanalyzed endpoints included hemolysis in the thawed sample and theadditional hemolysis which occurs upon 1:1 dilution with one of 2different diluent solution, PBS or isotype plasma.

In TABLE 6, the stability of the red blood cells following thaw is seenthe thaw hemolysis values and the beneficial effect of 4° C. incubationis observed in the dilution induced hemolysis values. Most important isthe ability of biochemical additives to enhance the protection duringthe protocol. The biochemical additives were used to stabilize the cellspost-thaw and demonstrate this effect by decreasing the dilution-induceshemolysis. Thus, the damage to the red blood cells duringcryopreservation is shown here to be both partially self-reversible andeither preventable or correctable with biochemical additions: Thetreatment conditions were as follows:

-   -   1. CPA only    -   2. CPA+50 mM Ribose    -   3. CPA+50 mM Ribose    -    50 mM Trehalose    -    500 μM Nifedipine    -    5 mM Pentoxifylline

 50 μg/ml Flurbiprofen TABLE 6 Biochemical Stabilization AgainstPost-Thaw Hemolysis Immediate 4° C. Condition PBS Plasma PBS Plasma CPAOnly Thaw (%) 2.15 2.23 Dilution (%) 6.39 5.57 4.55 3.73 CPA + RiboseThaw (%) 1.62 2.05 Dilution (%) 8.94 6.60 5.85 3.85 CPA + All Thaw (%)2.71 2.56 Additives Dilution (%) 3.53 3.23 2.62 2.26

EXAMPLE 7

The following experiment demonstrates the ability of alternativecryoprotectants at low, transfusable concentrations to act incombination to enhance protection against freeze/thaw induced hemolysis.

A single packed red blood cell unit containing ADSOL was obtained andprepared using standard blood banking protocols. The ADSOL was removedby centrifugation and a volume of preservation solution was added. Avolume of the resulting red blood cell unit was mixed with an equalvolume of preservation solution containing the desiredcryoprotectant(s). The final concentrations of cryoprotectants used were7.5% dextran (Dex; 40,000 MW), 2% polyvinyl pyrrolidone (PVP; 40,000 MW)and 5% hydroxyethyl starch (HES). The red blood cell samples containingcryoprotectants were submerged in liquid nitrogen for three minutes tofreeze and immediately transferred to a −80° C. freezer for extendedstorage. Following storage for 3-7 days, the samples were thawed at 37°C. in a water bath, and the level of thaw hemolysis was determined asdescribed as the ratio of free hemoglobin to total hemoglobin. Thelevels of thaw hemolysis are shown in TABLE 7 below: TABLE 7 LEVEL OFTHAW HEMOLYSIS % Hemolysis Dex 11.19 ± 0.38 PVP 47.39 ± 2.42 HES 43.17 ±10.1 Dex/PVP  6.42 ± 1.38 Dex/HES  8.56 ± 0.82 PVP/HES 23.46 ± 4.84Dex/PVP/HES  4.44 ± 0.04

This example confirms that extremely low, transfusable concentrations ofcryoprotectants can work in combination to protect red blood cellsduring cryopreservation better than any single cryoprotectant alone.

In view of the above disclosure, one of ordinary skill in the art shouldunderstand and appreciate that one illustrative embodiment of thepresent invention includes a red blood cell storage composition whichincludes a composition of red blood cells and biochemistry alteringreagents, the biochemistry altering reagents being present at aconcentration so as to reduce the percent hemolysis of the red bloodcells during the freeze-thaw cycle below that of the percent hemolysisof the red blood cells in the absence the biochemistry alteringreagents. Preferably the reagents of the present illustrative embodimentare selected from: modifiers of glycolytic/metabolic components,modifiers of antioxidant potential, effectors of intracellular ionicdistribution, modifiers of membrane fluidity, modifiers of cytoskeletalstructure, effectors of the cyclooxygenase second messenger pathway,effectors of the lipoxygenase second messenger pathway, effectors of thehexose monophosphate second messenger pathway, effectors of thephosphorylation second messenger pathway, modifiers of the cyclicnucleotide regulated second messenger pathway, and combinations thereof.More preferably, the reagents of the present illustrative embodiment areselected such that: the modifier of glycolytic/metabolic components isselected from glucose, pyruvate, inorganic phosphate, products of theglycolytic pathway such as adenosine triphosphate, nicotinamide adeninedinucleotide, and inhibitors of glycolysis such as iodacetic acid,rotenone, and carbonyl cyanide p-(trifluoromethoxyl)-phenyl hydrazine;the modifier of antioxidant potential is selected from glutathione,tocopherol, ascorbate, α-tocopherol, mannitol, bioflavonoids, andderivatives of bioflavonoids including rutin, quercetin, and curcumin;the effector of intracellular ionic is distribution acting through thesecond messenger pathway is selected from nifedipine, nisoldipine,benzamil, glybenclamidediltiazem, clotrimizole, tetramethylammoniumdiphenylhydantoin, diisothiocyanatstilbene-2,2′-disulfonic acid,verapamil, amiloride, bumetamide,N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide and derivativesthereof; the modifier of membrane fluidity is selected frompentoxifylline, nicotinamide, amantadine, carnitine, palmitoylcarnitine, sphingosine, diethylnicotinamide, (nikethamide), nicotinicacid, trehalose, valinomycin, procaine, tetracaine, rimantadine,propanolol, and protease inhibitors such as leupeptin; the modifier ofcytoskeletal structure is selected from TAXOL, cytochalasins,paclitaxel, okadaic acid and polyamines such as spermine, spermidine, orputrecine; the effector of the cyclooxygenase and lipoxygenase secondmessenger pathways is selected from flurbiprofen, dipyridamole,salicyclic acid, and indomethacin; the effector of the hexosemonophosphate second messenger pathway is selected from ribose orpyruvate; the effector of the phosphorylation second messenger pathwayis selected from H7 (1-[5-isoquinolinylsulfonyl]-2-methylpiperazine),staurosporin, chelerythrine, and diacylglycerol palmitoyl carnitine; andthe modifier of the cyclic nucleotide regulated second messenger pathwayis selected from adenosine, sodium nitroprusside, dibutyl AMP, dibutylGMP.

The illustrative composition may also be formulated such that thebiochemistry altering reagents are combined with one or morecryoprotective agent. In one preferred embodiment, the cryoprotectiveagent may be a permanent cryoprotectant, preferably the permanentcryoprotectant may be selected from glycerol, dimethyl sulfoxide,2,3-butandiol, 1,2-propandiol, 1,3-propandiol, polypropylene glycol,N,N-dimentylacetamide, and 1,3,5-trimethyl pentanetriol and combinationsthereof. Alternatively, the composition may be formulated such that thecryoprotective agent is a non-permanent cryoprotectant, which preferablymay be selected from hydoxyethyl starch, dextran, polyvinyl pyrrolidone,polyethylene, polyethylene glycol and combinations thereof. Acombination of one or more permanent cryoprotectants and one or morenon-permanent cryoprotectants may also be utilized in the formulation ofthe present illustrative embodiment. When the formulation includes anon-permanent cryoprotectant it is preferred that the non-permanentcryoprotectant have a molecular weight of at least about 5,000 MW.

Another illustrative embodiment of the present invention includes ahuman red blood cell composition including: red blood cells; a storagecomposition; and a composition of reagents, wherein the composition ofreagents is selected from: modifiers of metabolic components, modifiersof antioxidant potential, effectors of intracellular ionic distribution,modifiers of membrane fluidity, modifiers of cytoskeletal structure,effectors of the cyclooxygenase second messenger pathway, effectors ofthe lipoxygenase second messenger pathway, effectors of the hexosemonophosphate second messenger pathway, effectors of the phosphorylationsecond messenger pathway, modifiers of the cyclic nucletide regulatedsecond messenger pathway, and combinations thereof.

In such an illustrative composition, it is preferred that: the modifierof glycolytic/metabolic components is selected from glucose, pyruvate,inorganic phosphate, products of the glycolytic pathway such asadenosine triphosphate, nicotinamide adenine dinucleotide, andinhibitors of glycolysis such as iodacetic acid, rotenone, and carbonylcyanide p-(trifluoromethoxyl)-phenyl hydrazine; the modifier ofantioxidant potential is selected from glutathione, tocopherol,ascorbate, α-tocopherol, mannitol, bioflavonoids, and derivatives ofbioflavonoids including rutin, quercetin, and curcumin; the effector ofintracellular ionic distribution acting through the second messengerpathway is selected from nifedipine, nisoldipine, benzamil,glybenclamidediltiazem, clotrimizole, tetramethylammoniumdiphenylhydantoin, diisothiocyanatstilbene-2,2′-disulfonic acid,verapamil, amiloride, bumetamide,N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide and derivativesthereof; the modifier of membrane fluidity is selected frompentoxifylline, nicotinamide, amantadine, carnitine, palmitoylcarnitine, sphingosine, diethylnicotinamide, (nikethamide), nicotinicacid, trehalose, valinomycin, procaine, tetracaine, rimantadine,propanolol, and protease inhibitors such as leupeptin; the modifier ofcytoskeletal structure is selected from TAXOL, cytochalasins,paclitaxel, okadaic acid and polyamines such as spermine, spermidine, orputrecine; the effector of the cyclooxygenase and lipoxygenase secondmessenger pathways is selected from flurbiprofen, dipyridamole,salicyclic acid, and indomethacin; the effector of the hexosemonophosphate second messenger pathway is selected from ribose orpyruvate; the effector of the phosphorylation second messenger pathwayis selected from H7 (1-[5-isoquinolinylsulfonyl]-2-methylpiperazine),staurosporin, chelerythrine, and diacylglycerol palmitoyl carnitine; andthe modifier of the cyclic nucleotide regulated second messenger pathwayis selected from adenosine, sodium nitroprusside, dibutyl AMP, dibutylGMP.

The illustrative composition may be formulated such that the compositionof reagents are combined with one or more cryoprotective agents. In onesuch illustrative embodiment, the cryoprotective agent is a permanentcryoprotectant selected from glycerol, dimethyl sulfoxide,2,3-butandiol, 1,2-propandiol. 1,3-propandiol, polypropylene glycol,N,N-dimentylacetamide, 1,3,5-trimethyl pentanetriol and combinationsthereof. In another such illustrative embodiment, the cryoprotectiveagent is a non-permanent cryoprotectant selected from hydoxyethylstarch, dextran, polyvinyl pyrrolidone, polyethylene, polyethyleneglycol and combinations thereof. Preferably the cryoprotective solutionis a combination of one or more permanent cryoprotectants and one ormore non-permanent cryoprotectants. When a non-permanent cryoprotectantis utilized in the formulation, it is preferred that the non-permanentcryoprotectant have a molecular weight of at least 5000 MW.

The illustrative embodiment should be formulated such that thebiochemical reagents are present in a concentration so that upon invitro preservation of the red blood cells at a temperature below the redblood cells normal physiological temperature for a period of about 2-7days, the red blood cells exhibit decreased hemolysis during afreeze-thaw cycle and increased in vitro functional activity whencompared to red blood cells preserved under the same conditions but inthe absence of the biochemical reagents. Thus in one preferredembodiment of the present illustrative embodiment the nifedipine has aconcentration of about 500 μM, the cytochalasin B has a concentration ofabout 20 μM, the Taxol has a concentration of about 500 μM, thepentoxifylline has a concentration of about 5 mM, the flurbiprofen has aconcentration of about 25 μg/ml, the nikethamide has a concentration ofabout (1% v/v), the ribose has a concentration of about 0.50 mM, and thetrehalose has a concentration of about 50 mM.

It should also be appreciated that the present invention includes amethod for storing human red blood cells including: (a) isolating redblood cells from fresh human blood; (b) forming a red blood cellcomposition; and (c) storing the resulting red blood cell composition ata temperature below normal human physiological temperature effective topreserve the blood cells for a selected period of time. Preferably thered blood cell composition should be formulated so as to include: redblood cells; a storage composition; and a composition of reagents. Thecomposition of reagents should be selected from: modifiers of metaboliccomponents, modifiers of antioxidant potential, effectors ofintracellular ionic distribution, modifiers of membrane fluidity,modifiers of cytoskeletal structure, effectors of the cyclooxygenasesecond messenger pathway, effectors of the lipoxygenase second messengerpathway, effectors of the hexose monophosphate second messenger pathway,effectors of the phosphorylation second messenger pathway, modifiers ofthe cyclic nucleotide regulated messenger pathway, and combinationsthereof. In practicing the present illustrative method, the temperaturebelow normal physiological temperature effective to preserve the redblood cells for selected period of time is preferably about −10° C. to−193° C., and more preferably is about −80° C.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept and scopeof the invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the concept and scope of the invention as defined by the appendedclaim.

1.-7. (canceled)
 8. A red blood cell composition comprising: (a) redblood cells; (b) a combination of biochemistry altering reagentsselected from the group consisting of pentoxyfylline, flurbiprofen,nicotinamide, nikethamide, nifedipine, amiloride, taxol, cytochalasin B,trehalose, ribose, a protease inhibitor, a bioflavonoid, andbioflavonoid derivative; and (c) one or more cryoprotective agents. 9.The composition of claim 8, further comprising a storage composition.10. The composition of claim 8, wherein the protease inhibitor isleupeptin.
 11. The composition of claim 8, wherein the bioflavinoidderivative is selected from the group consisting of rutin, quercetin,and curcumin.
 12. The composition of claim 8, wherein the one or morecryoprotective agents is selected from the group consisting of glycerol,dimethyl sulfoxide, 2,3 butanediol, 1,2-propanediol, 1,3-propanediol,polypropylene glycol, N,N-dimethylacetamide, and 1,3,5-trimethylpentanetriol.
 13. The composition of claim 8, wherein the one or morecryoprotective agents is selected from the group consisting ofhydroxyethyl starch, dextran, polyvinyl pyrrolidone, polyethylene, andpolyethylene glycol.
 14. The composition of claim 8, wherein the one ormore cryoprotective agents comprise a combination of one or more cellmembrane penetrating cryoprotectants and one or more cryoprotectantsthat do not penetrate cell membranes.
 15. The composition of claim 14,wherein the one or more cryoprotectants that do not penetrate cellmembranes have molecular weights of at least 5,000 MW.
 16. Thecomposition of claim 8, comprising pentoxyfylline, flurbiprofen,nikethamide, nifedipine, taxol, cytochalasin B, trehalose, and ribose.17. The composition of claim 8, wherein the nifedipine has aconcentration of about 500 μM, the cytochalasin B has a concentration ofabout 20 μM, the pentoxifylline has a concentration of about 5 mM, theflurbiprofen has a concentration of about 25 μg/ml, the nikethamide hasa concentration of about 1% (v/v), the ribose has a concentration ofabout 50 mM, and the trehalose has a concentration of about 50 mM. 18.The composition of claim 8, wherein the biochemistry altering reagentsare present in a concentration so that upon in vitro preservation of thered blood cells at a temperature at or below the red blood cells'physiological temperature for a period of about 2-7 days, the red bloodcells exhibit decreased hemolysis during a freeze-thaw cycle andincreased in vitro functional activity when compared to red blood cellspreserved under the same conditions but in the absence of thebiochemistry altering reagents.
 19. A method for the prolonged storageof red blood cells comprising creating the composition of claim 8 andstoring the composition at a temperature below the red blood cells'physiological temperature.
 20. The method of claim 19, wherein thetemperature below the red blood cells' physiological temperature is fromabout −10° C. to about −193° C.
 21. The method of claim 19, wherein thetemperature below the red blood cells' physiological temperature isabout −80° C.
 22. The method of claim 12, wherein the red blood cellsare isolated from human blood prior to creating the composition.